Histological Analysis of a Mouse Model of the 22q11.2 Microdeletion Syndrome

Figure 1. The radial migration of cortical excitatory neurons in Del(3.0Mb)/+ embryos was indistinguishable from those of WT. (A) Experimental design of in utero electroporation. The plasmid mix containing pCAG-turboRFP (0.5 μg/μL) and pCAG-H2B-EGFP (0.3 μg/μL) was electroporated into the littermates that resulted from crossing between a male Del(3.0Mb)/+ and a female wild type (WT) mouse at embryonic day 14 (E14), and then fixed at E18. (B) Electroporated neurons of Del(3.0Mb)/+ embryos migrated mostly into the top of the cortical plate, which was not indistinguishable from those of WT. The slices of electroporated brains were stained with DAPI (nuclei, blue). The fluorescent signals of turboRFP (red, cytosol) and EGFP (green, nuclei) were merged. High magnification images of individual migrating neurons are shown in the inlets. Scale bar: 100 μm. (C) Histogram of GFP+ cell position in the cortical thickness. The thickness from the border between intermediate zone and ventricular zone to the surface of the brain was divided into 5 bins. The percentages of the GFP+ nuclei in each bin were evaluated. The bars represent the mean ± SD. Two-way ANOVA was conducted (WT: 673 cells from 3 brains,…

Figure 2. Gross histological organization of embryonic Del(3.0Mb)/+ mice is normal. (A) Layer structure of the E18 cerebral cortex. Coronal sections at the somatosensory area were stained with TAG1 (green), Ctip2 (red), and DAPI (blue). Scale bar: 200 μm. (B) Quantification of the thickness of layer II–IV (from the upper surface of Ctip2-labeled sector to the upper surface of DAPI dense sector), layer V–VI (Ctip2-labeled sector), and IZ–VZ (from the border between Ctip2 and TAG1 labeled sectors to the ventricular surface of the pallium). The thickness of each sector was not significantly changed in Del(3.0Mb)/+ (2-sided unpaired t-test with Welch’s correction, layer II–IV; p = 0.9364, layer V–VI; p = 0.0881, IZ–VZ; p = 0.6593, WT: 4 brains, Del: 9 brains). IZ, intermediate zone; VZ, ventricular zone. (C) No gross differences in the axonal tracts in the E18 forebrains were found. Sections of indicated genotypes at the level of somatosensory area were stained with anti-TAG1 (green) and anti-L1 (red) antibodies, and DAPI (blue). CC, corpus callosum. Scale bar: 500 μm. (D) Quantification of the thickness of CC. The thickness of CC at the midline was not significantly altered between WT and…

Figure 3. Gross cortical layer structure of adult Del(3.0Mb)/+ is preserved. (A) Coronal sections of the adult (6–12 months old) brains at the somatosensory area were stained with anti-Cux1 (green) and anti-Ctip2 (red) antibodies, and DAPI (blue). Scale bar: 300 μm. (B) Quantification of the thickness of layer II–IV (Cux1 heavily labeled sector), layer V (Ctip2 heavily labeled sector), layer VI (Ctip2 weakly labeled sector), and the white matter (WM, below layer VI). No significant changes in the thickness of any cortical layers and the WM (2-sided unpaired t-test with Welch’s correction, layer II–IV: p = 0.2959, layer V: p = 0.6645, layer VI: p = 0.1943, WM: p = 0.3813, WT: 5 brains, Del: 6 brains). WM, white matter. Horizontal bars in scatter plots (B) represent mean ± SD.

Figure 4. Dendritic arbors of subsets of neurons in adult Del(3.0Mb)/+ are slightly but significantly reduced. (A) Experimental design for visualization of individual neurons using EGFP-expressing AAV vector. About 50 µL of AAV solution in low titer (1011 virus genome (vg)/mL) was administered to adult (6 months old) mice via the retro-orbital route. These mice were fixed 7 days after the injection. Representative image of a sagittal section of the WT brain was shown (right). Scale bar: 500 μm. (B–E) Representative images of GFP+ individual neurons in various brain regions of WT or Del(3.0Mb)/+. Scale bars: 50 μm. The complexity of dendrite branches was quantified with Sholl analysis (bottom of each panel). Error bars indicate ± SD. The red lines and blue lines in the graphs represent WT and Del(3.0Mb)/+, respectively. The number of intersections in a range of 20–100 μm in the WT and Del(3.0Mb)/+ mouse brains were analyzed with two-way ANOVA (distance from soma and genotypes). The dendrite branches were significantly reduced in Del(3.0Mb)/+ medial prefrontal cortex (mPFC) (C) and nucleus accumbens (NAc) (E), while no significant changes were observed in the somatosensory area (S1) (B) and…

Figure 5. Dendritic spine densities of subsets of neurons in Del(3.0Mb)/+ are reduced. Dendritic spine density was evaluated on Golgi–Cox impregnated brain slices of adult (12 months old) WT or Del(3.0Mb)/+ mice. Representative images of spines visualized with Golgi–Cox staining are shown on the right (A,D–F). In accordance with the results of AAV-labeled neurons, the spine density (spine number/μm) of basal dendrites of pyramidal neurons in layer V in the somatosensory area (A) was significantly reduced, while those of medial spiny neurons in the Str (B) and in NAc (C) were not. In addition, the spine densities of basal dendrites of pyramidal neurons in layer II/III of somatosensory area (D) and those in layer V of mPFC (E) were reduced, while the significance was not detected in the first branch of apical dendrite of CA1 pyramidal neurons in the hippocampus. Two-sided unpaired t-tests with Welch’s correction were conducted. (A) p = 0.0022, ** p < 0.01, WT: 18 dendrites from 3 brains, Del: 17 dendrites from 3 brains. (B) p = 0.0665, WT: 12 dendrites from 3 brains, Del: 14 dendrites from 3 brains. (C) p = 0.8544, WT: 18 dendrites from 3 brains, Del: 21 dendrites from 3 brains. (D) p =…

Figure 6. Axon innervation of dopaminergic neurons into the mPFC in Del(3.0Mb)/+ is reduced. (A) Gross axonal tracts of dopaminergic neurons were unaffected. Sagittal sections of WT or Del(3.0Mb)/+ mouse brains were stained with anti-tyrosine hydroxylase (TH, red) antibody and DAPI (blue). SNpc, substantia nigra pars compacta. VTA, ventral tegmental area. VTA is located in different sagittal planes. Scale bar: 1 mm. (B) TH axons in the prefrontal area are reduced in Del(3.0Mb)/+ mice. Coronal sections of prefrontal area were stained with anti-TH (green) and anti-Ctip2 (red) antibodies. The right margin of the tissue section corresponds to the midline of the brain. Scale bar: 300 μm. (C) Quantification of the density of TH axons. Two-sided unpaired t-test with Welch’s correction, WT vs. Del; p = 0.0397, * p < 0.05, WT: 7 brains, Del: 8 brains (6–12 months old). Horizontal bars in scatter plots (C) represent mean ± SD.